<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div class="">Hi Vishaka & Cindi,</div><div class=""><span class="Apple-tab-span" style="white-space:pre"> </span>If the autofocus is set up to find focus, then shift to a user-defined defocus, you could end up at a high defocus without realizing it. Basically, for cryo specimens near focus, the image features are not strong compared to the noise, so autofocus sometimes gives the best cross-corellation on the noise, which can result is a random defocus. One test is to set the user-defined defocus at zero, then manually take repeated autofocus readings. The result of these should converge—usually by over-or under-shooting true focus, then oscillating about focus with a decreasing amplitude. I wrote a little article about this in Microscopy Today, and it applies to all the autofunctions I tested.</div><div style="orphans: 2; widows: 2;" class=""><span class="Apple-tab-span" style="white-space: pre;"> </span>Yours,</div><div style="orphans: 2; widows: 2;" class=""><span class="Apple-tab-span" style="white-space: pre;"> </span>Bill</div><div class=""><br class=""></div><div><blockquote type="cite" class=""><div class="">On Mar 12, 2018, at 3:52 PM, Schwartz, Cindi (NIH/NIAID) [E] <<a href="mailto:cindi.schwartz@nih.gov" class="">cindi.schwartz@nih.gov</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div class="WordSection1" style="page: WordSection1; font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;"><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Hi Vishaka,<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Last week, we noticed a similar phenomenon on our newly installed Falcon III. We collected data with the Falcon III on our Krios using both EPU and manual collection via TIA. (Long exposures, many fractions, multiple magnifications) In our case, we saw that the thon rings weren’t missing per se, but we were getting extremely high defocus (~20 um defocus on some images) and so we couldn’t see the thon rings out to the edge. In the software, we have the focus set at -1 to -3. So we tried collecting manual images and got similar results (used EPU to switch states to ease imaging). We are positive we are at eucentric height and are at focus when we start. We checked using the diffraction pattern before imaging in nearby regions on the same ‘flat’ grid square.<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">I wonder if there is a bug somewhere in the software/hardware because your image looks very underfocus. Could it be that instead of the normal defocus you think you should be getting, you are just getting some crazy defocused image?<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Are other people experiencing this? Is it something to do with the autofocus routine or the amount of signal in a focus area needed for cross correlation? Scope alignments?<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Thanks,<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Cindi<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 10.5pt; font-family: 'Century Gothic', sans-serif;" class="">-- </span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: 'Lucida Handwriting';" class="">Cindi L. Schwartz</span><span style="font-size: 12pt; font-family: Arial, sans-serif;" class=""> </span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Electron Microscopist</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Rocky Mountain Labs/NIAID/NIH</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">903 South 4th Street</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">Hamilton, MT 59840</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class="">406-363-9228</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: Arial, sans-serif;" class=""><a href="mailto:Cindi.Schwartz@NIH.gov" target="_blank" style="color: rgb(149, 79, 114); text-decoration: underline;" class=""><span style="color: blue;" class="">Cindi.Schwartz@NIH.gov</span></a></span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: 'Iowan Old Style Roman'; color: rgb(155, 187, 89);" class=""><span id="cid:image001.png@01D3BA22.836BA280"><image001.png></span> Please remember our Earth before printing this email <span id="cid:image001.png@01D3BA22.836BA280"><image001.png></span></span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 13pt; font-family: 'Iowan Old Style Roman';" class="">*************************************************************************</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 12pt; font-family: 'Iowan Old Style Roman';" class="">The information in this e-mail and any of its attachments is confidential and may contain sensitive information. It should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage devices. National Institute of Allergy and Infectious Diseases shall not accept liability for any statements made that are sender's own and not expressly made on behalf of the NIAID by one of its representatives. </span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><span style="font-size: 13pt; font-family: 'Iowan Old Style Roman';" class="">*************************************************************************</span><span style="" class=""><o:p class=""></o:p></span></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><b class="">From:</b><span class="Apple-converted-space"> </span>Vishaka Santosh [<a href="mailto:vs4p@virginia.edu" style="color: rgb(149, 79, 114); text-decoration: underline;" class="">mailto:vs4p@virginia.edu</a>]<span class="Apple-converted-space"> </span><br class=""><b class="">Sent:</b><span class="Apple-converted-space"> </span>Friday, March 9, 2018 2:06 PM<br class=""><b class="">To:</b><span class="Apple-converted-space"> </span><a href="mailto:3dem@ncmir.ucsd.edu" style="color: rgb(149, 79, 114); text-decoration: underline;" class="">3dem@ncmir.ucsd.edu</a><br class=""><b class="">Subject:</b><span class="Apple-converted-space"> </span>[3dem] Issue with Thon rings<o:p class=""></o:p></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div><div class=""><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Hello everyone! I am having a rather bizarre issue when analyzing my data. It was collected at 96kC with an exposure of 3.01 seconds for 78 fractions. Using MotionCor2, I attempted to analyze the quality of the data and when I look at the Thon rings, they are either very weak or not present. Any one have any ideas what could be wrong? I didn't collect this on a phase plate and it was collected on a Titan Krios at 300kV<br class=""><span id="cid:image004.jpg@01D3BA22.83FF08E0"><image004.jpg></span><span id="cid:image005.jpg@01D3BA22.83FF08E0"><image005.jpg></span><br clear="all" class=""><o:p class=""></o:p></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class=""><o:p class=""> </o:p></div></div><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">--<span class="Apple-converted-space"> </span><o:p class=""></o:p></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">Vishaka Santosh<o:p class=""></o:p></div><div class=""><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">UVA Class of 2011, BS in Chemistry with a specialization in Biochemistry<o:p class=""></o:p></div></div></div><div class=""><div style="margin: 0in 0in 0.0001pt; font-size: 11pt; font-family: Calibri, sans-serif;" class="">VCU Class of 2013, MS in Biochemistry <o:p class=""></o:p></div></div></div></div></div></div><span style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; float: none; display: inline !important;" class="">_______________________________________________</span><br style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class=""><span style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; float: none; display: inline !important;" class="">3dem mailing list</span><br style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class=""><a href="mailto:3dem@ncmir.ucsd.edu" style="color: rgb(149, 79, 114); text-decoration: underline; font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class="">3dem@ncmir.ucsd.edu</a><br style="font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class=""><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem" style="color: rgb(149, 79, 114); text-decoration: underline; font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px;" class="">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a></div></blockquote></div><div class=""><span class="Apple-style-span" style="border-collapse: separate; font-variant-ligatures: normal; font-variant-position: normal; font-variant-numeric: normal; font-variant-alternates: normal; font-variant-east-asian: normal; line-height: normal; border-spacing: 0px;"><div class=""><br class=""></div></span><br class="Apple-interchange-newline">
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